The accurate and rapid detection of viral and bacterial pathogens in human patients and populations is of critical medical and epidemiologic importance. Historically, diagnostic techniques have relied on cell culture passaging and various immunological assays or staining procedures. Accurate and sensitive detection of infectious disease agents is still difficult today, despite a long history of progress in this area. Traditional methods of culture and antibody-based detection still play a central role in microbiological laboratories despite the problems of the delay between disease presentation and diagnosis, and the limited number of organisms that can be detected by these approaches. Faster diagnosis of infections would reduce morbidity and mortality, for example, through the earlier implementation of appropriate antimicrobial treatment. During the past few-decades, various methods have been proposed to achieve this; with those based on nucleic acid detection, including PCR and microarray-based techniques, seeming the most promising. In particular, PCR-based assays have been implemented, allowing for more rapid diagnosis of suspected pathogens with higher degree of sensitivity of detection. In clinical practice, however, the etiologic agent often remains unidentified, eluding detection in myriad ways. For example, some viruses are not amenable to culturing. At other times, a patient's sample may be of too poor quality or of insufficient titre for pathogen detection by conventional techniques. Moreover, both PCR- and antibody-based approaches may fail to recognize suspected pathogens simply due to natural genetic diversification resulting in alterations of PCR primer binding sites and antigenic drift.
DNA and oligonucleotide microarrays with the potential to detect multiple pathogens in parallel have been described (Wang et al. 2002; Urisman et al. 2005). However, unresolved technical questions prevent their routine use in the clinical setting. For example, how does one select the most informative probes for comprising a pathogen signature in light of amplification and cross-hybridization artifacts? What levels of fluorescent signal and signature probe involvement constitute a detected pathogen? What is the accuracy and sensitivity of an optimized detection algorithm? (Striebel et al. 2003; Bodrossy and Sessitsch, 2004; Vora et al. 2004).
Accordingly, there is a need in this field of technology for alternative and improved methods of detection of nucleic acids. In particular, there is a need for alternative and/or improved diagnostic methods for the detection of pathogens.